High-Throughput Cell-Based Screening Methodology For Evaluating Carbohydrate-Active Enzymes

Abstract

The present disclosure relates, in one aspect, to the discovery of a high throughput screening (HTS) method to rapidly screen for GH/GS variants that are generated using directed evolution techniques and that can significantly enhance glycosynthase catalytic activity or product specificity.

What is claimed: 
     
         1 . A method of determining if a protein has transglycosylase activity, the method comprising:
 contacting the protein with an azido glycosyl donor and a glycosyl acceptor to form a system, and   measuring any change in azide concentration in the system.   
     
     
         2 . The method of  claim 1 , wherein the azido glycosyl donor is substituted with an azido group at an anomeric or non-anomeric carbon. 
     
     
         3 . The method of  claim 1 , wherein the azide concentration of the system comprises an inorganic azide and an anomeric glycosyl azide species or a non-anomeric glycosyl azide species. 
     
     
         4 . The method of  claim 1 , wherein the measuring step comprises contacting the system with a reagent comprising a strained alkyne coupled to a dye, under conditions that allow for reaction of the strained alkyne with any azide or azido compound present in the system. 
     
     
         5 . The method of  claim 4 , wherein the reagent comprises bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctyne (DBCO), or any other strained alkyne. 
     
     
         6 . The method of  claim 4 , wherein the reagent comprises 5-carboxytetramethylrhodamine (5-TAMRA), 6-carboxytetramethylrhodamine (6-TAMRA), or any combinations thereof. 
     
     
         7 . The method of  claim 4 , wherein the strained alkyne and the dye are covalently linked by a linker in the reagent. 
     
     
         8 . The method of  claim 7 , wherein the linker comprises a polyethylene glycol linker. 
     
     
         9 . The method of  claim 1 , wherein the measuring step uses as a control a protein that has no measurable transglycosylase activity or has a known transglycosylase activity. 
     
     
         10 . The method of  claim 1 , wherein the protein is a mutated glycosyl hydrolase (GH). 
     
     
         11 . The method of  claim 1 , wherein the protein is expressed in a cell. 
     
     
         12 . The method of  claim 11 , wherein the cell comprises  E. coli  or  Pichia pastoris.    
     
     
         13 . The method of  claim 11 , wherein the system is within the cell (intracellular). 
     
     
         14 . The method of  claim 4 , wherein the measuring step comprises monitoring fluorescence of the system. 
     
     
         15 . The method of  claim 13 , wherein fluorescence activated cell sorting (FACS) is used to separate individual cells by measured fluorescence. 
     
     
         16 . The method of  claim 15 , which is configured for high-throughput screening. 
     
     
         17 . A polypeptide comprising an amino acid sequence of SEQ ID NO:1,
 wherein the polypeptide comprises the mutation D224G (SEQ ID NO:2) with respect to SEQ ID NO:1,   wherein the polypeptide further comprises at least one additional mutation selected from the group consisting of L15K, N70D, A366V, T392S, K395N, D400A, T413P, I428T, and T429P.   
     
     
         18 . The polypeptide of  claim 17 , wherein the at least one additional mutation to an amino acid sequence of SEQ ID NO:2 is selected from the group consisting of: L15K-N70D; N70D-T392S; N70D-T392S-A366V-K395N; N70D-T392S-D400A; N70D-T392S-I428T; N70D-D400A-T413P-T429P. 
     
     
         19 . The polypeptide of  claim 18 , which is selected from the group consisting of SEQ ID NOs:3-8.